The long term goal of our research is to find new ways to prevent or to treat the infection caused by respiratory syncytial virus (RSV). In order to reach this goal, we will examine the molecular aspects by which the virus enters its natural targets, in particular, human bronchial epithelial cells. The route used by RSV to infect its primary target cells has not been clearly established. The current proposal would help to clarify the influence of the cell's population context and cellular state during RSV infection and take them into account when we test endocytic pathways using approaches providing down and up phenotypes regarding RSV infection in NHBE cells and well-differentiated bronchial epithelial cell cultures. The specific aims of this project are: (1) Determine if the cell's populaion context exerts an influence on the RSV infection and entry in undifferentiated primary cultures of normal human bronchial epithelial (NHBE) cells. Using high cell content screening, the influence that several features associated with a cell's population context and cellular state have on RSV infection and entry will be evaluated. (2) Determine the entry pathway that RSV hijacks to infect undifferentiated NHBE cells by taking cell's population context into account. Experiments will be designed using pharmacological (small molecules) and genetic approaches that generate either down or up phenotypes in each of the different endocytic pathways. Modulator-induced changes on cell's population context will be characterized to correct their effects when evaluating their positive or negative impact on RSV entry, fusion or infection in order to identify those interventions, and consequent endocytic pathway, involved in RSV infection. (3) Determine RSV entry pathway in well-differentiated bronchial epithelial cell (BEC) cultures. Using the general down or up assay approach, several endocytic pathways will be probed through pharmacological interventions to determine RSV entry route in well-differentiated BEC. For the pharmacological interventions, we will follow the general guidelines described in Aim 2, and the impact of these interventions on RSV infection and entry will be evaluated taking into consideration the potential effect on the state of the cells (i.e. cilia status and apoptosis). In Colombia, RSV is the most important pathogen agent causing 41.8% of the new cases of lower tract respiratory illness in infants. Understanding the mechanistic process of the RSV infection will help in the development of the vaccines and therapies. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page